Phase I Study of Dalteparin in Combination With Sunitinib in Patients With Metastatic Clear Cell Renal Carcinoma.

BACKGROUND: Based on the tumor-driven concomitant activation of angiogenesis and coagulation we conducted a phase I combination study of sunitinib with the low molecular weight heparin dalteparin in patients with metastatic clear cell renal cell carcinoma (ccRCC). MATERIALS AND METHODS: Patients received standard treatment with sunitinib (50 mg daily, 4 weeks on, 2 weeks off). During the second week of no sunitinib in the first cycle (week 6) patients received dalteparin monotherapy (in escalating doses). Combination therapy of the 2 agents was administered from the second cycle onward. Seventeen patients were enrolled at 3 dose levels of dalteparin. RESULTS: Diarrhea and fatigue were the most frequent reported drug-related toxicities (41%). One dose-limiting toxicity (grade 3 anemia) was observed at the highest dose level of dalteparin. There were 4 partial responses (24%) and the median progression-free survival in this study was 14 months (95% confidence interval, 8.0-23.4). Anti-factor Xa levels were increased during combination therapy compared with dalteparin monotherapy. CONCLUSIONS: Combination therapy of sunitinib with therapeutic doses of dalteparin is safe and well tolerated. The increased anti-factor Xa levels during combination treatment suggest that sunitinib might increase the anticoagulation activity of dalteparin. The positive safety profile warrants prospective evaluation of the clinical benefit of this combination strategy in patients with ccRCC.

Combination of the histone deacetylase inhibitor vorinostat with bevacizumab in patients with clear-cell renal cell carcinoma: a multicentre, single-arm phase I/II clinical trial.

BACKGROUND: Class II histone deacetylase (HDAC) inhibitors induce hypoxia-inducible factor-1 and -2α degradation and have antitumour effects in combination with vascular endothelial growth factor (VEGF) inhibitors. In this study, we tested the safety and efficacy of the HDAC inhibitor vorinostat and the VEGF blocker bevacizumab in metastatic clear-cell renal cell carcinoma (ccRCC) patients previously treated with different drugs including sunitinib, sorafenib, axitinib, interleukin-2, interferon, and temsirolimus. METHODS: Patients with up to two prior regimens were eligible for treatment, consisting of vorinostat 200 mg orally two times daily × 2 weeks, and bevacizumab 15 mg kg-1 intravenously every 3 weeks. The primary end points were safety and tolerability, and the proportion of patients with 6 months of progression-free survival (PFS). Correlative studies included immunohistochemistry, FDG PET/CT scans, and serum analyses for chemokines and microRNAs. RESULTS: Thirty-six patients were enrolled, with 33 evaluable for toxicity and efficacy. Eighteen patients had 1 prior treatment, 13 patients had 2 prior treatments, and 2 patients were treatment naïve. Two patients experienced grade 4 thrombocytopenia and three patients had grade 3 thromboembolic events during the course of exposure. We observed six objective responses (18%), including one complete response and five partial responses. The proportion of patients with PFS at 6 months was 48%. The median PFS and overall survival were 5.7 months (confidence interval (CI): 4.1-11.0) and 13.9 months (CI: 9.8-20.7), respectively. Correlative studies showed that modulation of specific chemokines and microRNAs were associated with clinical benefit. CONCLUSIONS: The combination of vorinostat with bevacizumab as described is relatively well tolerated. Response rate and median PFS suggest clinical activity for this combination strategy in previously treated ccRCC.

Combination strategy targeting VEGF and HGF/c-met in human renal cell carcinoma models.

Alternative pathways to the VEGF, such as hepatocyte growth factor or HGF/c-met, are emerging as key players in tumor angiogenesis and resistance to anti-VEGF therapies. The aim of this study was to assess the effects of a combination strategy targeting the VEGF and c-met pathways in clear cell renal cell carcinoma (ccRCC) models. Male SCID mice (8/group) were implanted with 786-O tumor pieces and treated with either a selective VEGF receptor tyrosine kinase inhibitor, axitinib (36 mg/kg, 2×/day); a c-met inhibitor, crizotinib (25 mg/kg, 1×/day); or combination. We further tested this drug combination in a human ccRCC patient-derived xenograft, RP-R-01, in both VEGF-targeted therapy-sensitive and -resistant models. To evaluate the resistant phenotype, we established an RP-R-01 sunitinib-resistant model by continuous sunitinib treatment (60 mg/kg, 1×/day) of RP-R-01-bearing mice. Treatment with single-agent crizotinib reduced tumor vascularization but failed to inhibit tumor growth in either model, despite also a significant increase of c-met expression and phosphorylation in the sunitinib-resistant tumors. In contrast, axitinib treatment was effective in inhibiting angiogenesis and tumor growth in both models, with its antitumor effect significantly increased by the combined treatment with crizotinib, independently from c-met expression. Combination treatment also induced prolonged survival and significant tumor growth inhibition in the 786-O human RCC model. Overall, our results support the rationale for the clinical testing of combined VEGF and HGF/c-met pathway blockade in the treatment of ccRCC, both in first- and second-line setting.

Tasquinimod modulates suppressive myeloid cells and enhances cancer immunotherapies in murine models.

A major barrier for cancer immunotherapy is the presence of suppressive cell populations in patients with cancer, such as myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM), which contribute to the immunosuppressive microenvironment that promotes tumor growth and metastasis. Tasquinimod is a novel antitumor agent that is currently at an advanced stage of clinical development for treatment of castration-resistant prostate cancer. A target of tasquinimod is the inflammatory protein S100A9, which has been demonstrated to affect the accumulation and function of tumor-suppressive myeloid cells. Here, we report that tasquinimod provided a significant enhancement to the antitumor effects of two different immunotherapeutics in mouse models of cancer: a tumor vaccine (SurVaxM) for prostate cancer and a tumor-targeted superantigen (TTS) for melanoma. In the combination strategies, tasquinimod inhibited distinct MDSC populations and TAMs of the M2-polarized phenotype (CD206(+)). CD11b(+) myeloid cells isolated from tumors of treated mice expressed lower levels of arginase-1 and higher levels of inducible nitric oxide synthase (iNOS), and were less immunosuppressive ex vivo, which translated into a significantly reduced tumor-promoting capacity in vivo when these cells were coinjected with tumor cells. Tumor-specific CD8(+) T cells were increased markedly in the circulation and in tumors. Furthermore, T-cell effector functions, including cell-mediated cytotoxicity and IFNγ production, were potentiated. Taken together, these data suggest that pharmacologic targeting of suppressive myeloid cells by tasquinimod induces therapeutic benefit and provide the rationale for clinical testing of tasquinimod in combination with cancer immunotherapies.

Sunitinib dose escalation overcomes transient resistance in clear cell renal cell carcinoma and is associated with epigenetic modifications.

Sunitinib is considered a first-line therapeutic option for patients with advanced clear cell renal cell carcinoma (ccRCC). Despite sunitinib's clinical efficacy, patients eventually develop drug resistance and disease progression. Herein, we tested the hypothesis whether initial sunitinib resistance may be transient and could be overcome by dose increase. In selected patients initially treated with 50 mg sunitinib and presenting with minimal toxicities, sunitinib dose was escalated to 62.5 mg and/or 75 mg at the time of tumor progression. Mice bearing two different patient-derived ccRCC xenografts (PDX) were treated 5 days per week with a dose-escalation schema (40-60-80 mg/kg sunitinib). Tumor tissues were collected before dose increments for immunohistochemistry analyses and drug levels. Selected intrapatient sunitinib dose escalation was safe and several patients had added progression-free survival. In parallel, our preclinical results showed that PDXs, although initially responsive to sunitinib at 40 mg/kg, eventually developed resistance. When the dose was incrementally increased, again we observed tumor response to sunitinib. A resistant phenotype was associated with transient increase of tumor vasculature despite intratumor sunitinib accumulation at higher dose. In addition, we observed associated changes in the expression of the methyltransferase EZH2 and histone marks at the time of resistance. Furthermore, specific EZH2 inhibition resulted in increased in vitro antitumor effect of sunitinib. Overall, our results suggest that initial sunitinib-induced resistance may be overcome, in part, by increasing the dose, and highlight the potential role of epigenetic changes associated with sunitinib resistance that can represent new targets for therapeutic intervention.

Dll4 blockade potentiates the anti-tumor effects of VEGF inhibition in renal cell carcinoma patient-derived xenografts.

BACKGROUND: The Notch ligand Delta-like 4 (Dll4) is highly expressed in vascular endothelium and has been shown to play a pivotal role in regulating tumor angiogenesis. Blockade of the Dll4-Notch pathway in preclinical cancer models has been associated with non-productive angiogenesis and reduced tumor growth. Given the cross-talk between the vascular endothelial growth factor (VEGF) and Delta-Notch pathways in tumor angiogenesis, we examined the activity of a function-blocking Dll4 antibody, REGN1035, alone and in combination with anti-VEGF therapy in renal cell carcinoma (RCC). METHODS AND RESULTS: Severe combined immunodeficiency (SCID) mice bearing patient-derived clear cell RCC xenografts were treated with REGN1035 and in combination with the multi-targeted tyrosine kinase inhibitor sunitinib or the VEGF blocker ziv-aflibercept. Immunohistochemical and immunofluorescent analyses were carried out, as well as magnetic resonance imaging (MRI) examinations pre and 24 hours and 2 weeks post treatment. Single agent treatment with REGN1035 resulted in significant tumor growth inhibition (36-62%) that was equivalent to or exceeded the single agent anti-tumor activity of the VEGF pathway inhibitors sunitinib (38-54%) and ziv-aflibercept (46%). Importantly, combination treatments with REGN1035 plus VEGF inhibitors resulted in enhanced anti-tumor effects (72-80% growth inhibition), including some tumor regression. Magnetic resonance imaging showed a marked decrease in tumor perfusion in all treatment groups. Interestingly, anti-tumor efficacy of the combination of REGN1035 and ziv-aflibercept was also observed in a sunitinib resistant ccRCC model. CONCLUSIONS: Overall, these findings demonstrate the potent anti-tumor activity of Dll4 blockade in RCC patient-derived tumors and a combination benefit for the simultaneous targeting of the Dll4 and VEGF signaling pathways, highlighting the therapeutic potential of this treatment modality in RCC.

Inhibition of Hsp90 augments docetaxel therapy in castrate resistant prostate cancer.

First line treatment of patients with castrate resistant prostate cancer (CRPC) primarily involves administration of docetaxel chemotherapy. Unfortunately, resistance to docetaxel therapy is an ultimate occurrence. Alterations in androgen receptor (AR) expression and signaling are associated mechanisms underlying resistance to docetaxel treatment in CRPC. Heat shock protein 90 (Hsp90) is a molecular chaperone, which regulates the activation, maturation and stability of critical signaling proteins involved in prostate cancer, including the AR. This knowledge and recent advances in compound design and development have highlighted Hsp90 as an attractive therapeutic target for the treatment of CRPC. We recently reported the development of a MYC-CaP castrate resistant (MYC-CaP/CR) transplant tumor model, which expresses amplified wild type AR. Within, we report that a second generation Hsp90 inhibitor, NVP-AUY922, inhibits cell growth and significantly induces cell death in MYC-CaP/CR and Pten-CaP/cE2 cell lines. NVP-AUY922 induced proteasome degradation of AR, though interestingly does not require loss of AR protein to inhibit AR transcriptional activity. Further, NVP-AUY922 increased docetaxel toxicity in MYC-CaP/CR and Pten-CaP/cE2 cell lines in vitro. Finally, NVP-AUY922/docetaxel combination therapy in mice bearing MYC-CaP/CR tumors resulted in greater anti-tumor activity compared to single treatment. This study demonstrates that NVP-AUY922 elicits potent activity towards AR signaling and augments chemotherapy response in a mouse model of CRPC, providing rationale for the continued clinical development of Hsp90 inhibitors in clinical trials for treatment of CRPC patients.

Acquired tumor cell resistance to sunitinib causes resistance in a HT-29 human colon cancer xenograft mouse model without affecting sunitinib biodistribution or the tumor microvasculature.

Acquired resistance to anti-angiogenic tyrosine kinase inhibitors is an important clinical problem in treating various cancers. To what extent acquired resistance is determined by microenvironmental host-factors or by tumor cells directly is unknown. We previously found that tumor cells can become resistant to sunitinib in vitro. Here, we studied to what extent in vitro induced resistance of tumor cells determines in vivo resistance to sunitinib. In severe combined immunodeficient mice, tumors were established from HT-29 parental colon cancer cells (HT-29PAR) or the in vitro induced sunitinib resistant HT-29 cells (HT-29SUN). Treatment with sunitinib (40mg/kg/day) inhibited tumor growth of HT-29PAR tumors by 71±5%, while no inhibition of HT-29SUN tumor growth was observed. Intratumoral sunitinib concentrations and reduced MVD were similar in both groups. Ki67 staining revealed that tumor cell proliferation was significantly reduced with 30% in HT-29PAR tumors, but unaffected in HT-29SUN tumors upon sunitinib treatment. The lysosomal capacity reflected by LAMP-1 and -2 expression was higher in HT-29SUN compared to HT-29PAR tumors indicating an increased sequestration of sunitinib in lysosomes of resistant tumors. In conclusion, we demonstrate that tumor cells rather than host-factors may play a crucial role in acquired resistance to sunitinib in vivo.

Dietary protein restriction inhibits tumor growth in human xenograft models.

PURPOSE: Data from epidemiological and experimental studies suggest that dietary protein intake may play a role in inhibiting prostate and breast cancer by modulating the IGF/AKT/mTOR pathway. In this study we investigated the effects of diets with different protein content or quality on prostate and breast cancer. EXPERIMENTAL DESIGN: To test our hypothesis we assessed the inhibitory effect of protein diet restriction on prostate and breast cancer growth, serum PSA and IGF-1 concentrations, mTOR activity and epigenetic markers, by using human xenograft cancer models. RESULTS: Our results showed a 70% inhibition of tumor growth in the castrate-resistant LuCaP23.1 prostate cancer model and a 56% inhibition in the WHIM16 breast cancer model fed with a 7% protein diet when compared to an isocaloric 21% protein diet. Inhibition of tumor growth correlated, in the LuCaP23.1 model, with decreased serum PSA and IGF-1 levels, down-regulation of mTORC1 activity, decreased cell proliferation as indicated by Ki67 staining, and reduction in epigenetic markers of prostate cancer progression, including the histone methyltransferase EZH2 and the associated histone mark H3K27me3. In addition, we observed that modifications of dietary protein quality, independently of protein quantity, decreased tumor growth. A diet containing 20% plant protein inhibited tumor weight by 37% as compared to a 20% animal dairy protein diet. CONCLUSIONS: Our findings suggest that a reduction in dietary protein intake is highly effective in inhibiting tumor growth in human xenograft prostate and breast cancer models, possibly through the inhibition of the IGF/AKT/mTOR pathway and epigenetic modifications.

Development of a castrate resistant transplant tumor model of prostate cancer.

BACKGROUND: Currently, limited mouse models that mimic the clinical course of castrate resistant prostate development currently exist. Such mouse models are urgently required to conduct pre-clinical studies to assist in the understanding of disease progression and the development of rational therapeutic strategies to treat castrate resistant prostate cancer. METHODS: Wild type intact FVB male mice were injected by subcutaneous injection with Myc-CaP cells to establish androgen sensitive Myc-CaP tumors. Tumor bearing mice were castrated and resulting tumors serially passaged in pre-castrated FVB male mice to produce a bone fide Myc-CaP castrate resistant tumor. RESULTS: Immunohistochemical analysis revealed that initial androgen sensitive Myc-CaP tumors had strong nuclear transcriptional active androgen receptor expression, as indicated by marked c-MYC staining and were highly proliferative. Castration of tumor bearing animals resulted in cytoplasmic relocation of androgen receptor concurrent with loss of transcriptional activity and tumor proliferation. Serial passaging of castrate refractory Myc-CaP in pre-castrated male FVB mice resulted in the development of a bona fide castrate resistant Myc-CaP tumor which pheno-copied the original androgen sensitive parental Myc-CaP tumor. CONCLUSIONS: Developing a murine castrate transplant resistant tumor model that mimics the clinical course of human castrate resistant prostate cancer will create better opportunities to understand the development of castrate resistant prostate cancer and also allow for more rapid pre-clinical studies to stratify rational novel therapies for this lethal form of prostate cancer.

Concurrent HDAC and mTORC1 inhibition attenuate androgen receptor and hypoxia signaling associated with alterations in microRNA expression.

Specific inhibitors towards Histone Deacetylases (HDACs) and Mammalian Target of Rapamycin Complex 1 (mTORC1) have been developed and demonstrate potential as treatments for patients with advanced and/or metastatic and castrate resistant prostate cancer (PCa). Further, deregulation of HDAC expression and mTORC1 activity are documented in PCa and provide rational targets to create new therapeutic strategies to treat PCa. Here we report the use of the c-Myc adenocarcinoma cell line from the c-Myc transgenic mouse with prostate cancer to evaluate the in vitro and in vivo anti-tumor activity of the combination of the HDAC inhibitor panobinostat with the mTORC1 inhibitor everolimus. Panobinostat/everolimus combination treatment resulted in significantly greater antitumor activity in mice bearing androgen sensitive Myc-CaP and castrate resistant Myc-CaP tumors compared to single treatments. We identified that panobinostat/everolimus combination resulted in enhanced anti-tumor activity mediated by decreased tumor growth concurrent with augmentation of p21 and p27 expression and the attenuation of angiogenesis and tumor proliferation via androgen receptor, c-Myc and HIF-1α signaling. Also, we observed altered expression of microRNAs associated with these three transcription factors. Overall, our results demonstrate that low dose concurrent panobinostat/everolimus combination therapy is well tolerated and results in greater anti-tumor activity compared to single treatments in tumor bearing immuno-competent mice. Finally, our results suggest that response of selected miRs could be utilized to monitor panobinostat/everolimus in vivo activity.